Published Work

July 26, 2016

Transcription factor KLF2 regulates homeostatic NK cell proliferation and survival (Proc Natl Acad Sci USA. 113:5370)

Adoptive transfer of allogeneic natural killer (NK) cells into leukemia patients can lead to remission; however, therapies are hindered by inefficient expansion and limited persistence of these lymphocytes. We now report that Kruppel-like factor 2 (KLF2) regulates both NK cell proliferation and survival. KLF2 limits homeostatic expansion of NK cells in a cell-intrinsic manner. In addition, KLF2 instructs mature NK cells to home to IL-15–rich niches, which is necessary for continued survival under homeostatic conditions. Therefore, targeting KLF2 while providing rate-limiting survival factors such as transpresented IL-15 may improve NK cell engraftment and sustainability in cancer patients.

May 10, 2016

KLF2 is a rate-limiting transcription factor that can be targeted to enhance regulatory T cell production. (Proc Natl Acad Sci USA. 111:9579)

Regulatory T cells (Tregs) are crucial for preventing autoimmunity, and thus discovering an efficient means of generating antigen-specific Tregs is a medical priority. To this end, we demonstrate that transcription factor Krüppel-like factor 2 (KLF2) is necessary for the generation of antigen-induced Tregs and their in vivo counterpart, peripheral Tregs. Moreover, pharmaceutical drugs that stabilize KLF2 protein levels during the transition from CD4+CD25− T cell to CD4+CD25+FoxP3+ Treg augment production of these tolerizing lymphocytes. Results from this study indicate that KLF2 is a viable target for altering Treg development, which may significantly impact patients prescribed statins.

July 1, 2014

Follicular B cell trafficking within the spleen actively restricts humoral immune responses. (Immunity. 33:1-12)

August 5, 2010

Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.

Platelets regulate lymphatic vascular development through CLEC2-SLP-76 signaling. (Blood. 116:661)

March 24, 2010

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK–SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre–mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76–dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

Transcription factor KLF2 regulates the migration of naive T cells by restricting chemokine receptor expression patterns. (Nature Immunology. 9:292)

February 3, 2008

The migration patterns of naive and activated T cells are associated with the expression of distinct sets of chemokine receptors, but the molecular basis for this regulation is unknown. Here we identify Krupple-like factor 2 (KLF2) as a key transcriptional factor needed to prevent naive T cells from expressing inflammatory chemokine receptors and acquiring the migration patterns of activated T cells. Lineage-specific deletion of KLF2 resulted in fewer naive T cells in the blood and secondary lymphoid organs, whereas it expanded naive T cell numbers in nonlymphoid tissues; these effects were associated with altered expression of inflammatory chemokine receptors on naive T cells. KLF2 repressed the expression of several chemokine receptors, including CCR3 and CCR5. We thus conclude that KLF2 maintains proper T cell migration patterns by linking T cell movement and transcriptional regulation of chemokine receptor expression patterns.

Klf2 is an essential regulator of vascular hemodynamic forces in vivo. (Dev. Cell. 11:845)

January 25, 2025

Hemodynamic responses that control blood pressure and the distribution of blood flow to different organs are essential for survival. Shear forces generated by blood flow regulate hemodynamic responses, but the molecular and genetic basis for such regulation is not known. The transcription factor KLF2 is activated by fluid shear stress in cultured endothelial cells, where it regulates a large number of vasoactive endothelial genes. Here, we show that Klf2 expression during development mirrors the rise of fluid shear forces, and that endothelial loss of Klf2 results in lethal embryonic heart failure due to a high-cardiac-output state. Klf2 deficiency does not result in anemia or structural vascular defects, and it can be rescued by administration of phenylephrine, a catecholamine that raises vessel tone. These findings identify Klf2 as an essential hemodynamic regulator in vivo and suggest that hemodynamic regulation in response to fluid shear stress is required for cardiovascular development and function.

Syk and Slp-76 mutant mice reveal a cell autonomous hematopoietic cell contribution to vascular development. (Dev. Cell. 11:349)

September 5, 2006

Developmental studies support a common origin for blood and endothelial cells, while studies of adult angiogenic responses suggest that the hematopoietic system can be a source of endothelial cells later in life. Whether hematopoietic tissue is a source of endothelial cells during normal vascular development is unknown. Mouse embryos lacking the signaling proteins Syk and Slp-76 develop abnormal blood-lymphatic endothelial connections. Here we demonstrate that expression of GFPSlp-76 in a subset of hematopoietic cells rescues this phenotype, and that deficient cells confer focal vascular phenotypes in chimeric embryos consistent with a cell-autonomous mechanism. Endogenous Syk and Slp-76, as well as transgenic GFPSlp-76, are expressed in circulating cells previously proposed to be endothelial precursors, supporting a causal role for these cells. These studies provide genetic evidence for hematopoietic contribution to vascular development and suggest that hematopoietic tissue can provide a source of vascular endothelial progenitor cells throughout life.

Evidence for the requirement of ITAM domains but not SLP-76/Gads interaction for integrin signaling in hematopoietic cells. (Mol. Cell. Biol. 26:6936)

August 30, 2006

Syk tyrosine kinase and Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) are signaling mediators activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptors and integrins. While the signaling cascades descending from integrins are similar to immunoreceptors, the mechanism of Syk activation and SLP-76 recruitment remains unclear. We used an in vivo structure-function approach to study the requirements for the domains of Syk and SLP-76 in immunoreceptor and integrin signaling. We found that both SH2 domains and the kinase domain of Syk are required for immunoreceptor-dependent signaling and cellular response via integrins. While the Gads-binding domain of SLP-76 is needed for immunoreceptor signaling, it appears dispensable for integrin signaling. Syk and SLP-76 also are required for initiating and/or maintaining separation between the blood and lymphatic vasculature. Therefore, we correlated the signaling requirement of the various domains of Syk and SLP-76 to their requirement in regulating vascular separation. Our data suggest ITAMs are required in Syk-dependent integrin signaling, demonstrate the separation of the structural features of SLP-76 to selectively support immunoreceptor versus integrin signaling, and provide evidence that the essential domains of SLP-76 for ITAM signals are those which most efficiently support separation between lymphatic and blood vessels.

In vivo alpha1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines. (Genes and Development. 20:927)

Integrins are heterodimeric adhesion receptors associated with bidirectional signaling. In vitro studies support a role for the binding of evolutionarily conserved tyrosine motifs (NPxY) in the β integrin cytoplasmic tail to phosphotyrosine-binding (PTB) domain-containing proteins, an interaction proposed to be dynamically regulated by tyrosine phosphorylation. Here we show that replacement of both β1 integrin cytoplasmic tyrosines with alanines, resulting in the loss of all PTB domain interaction, causes complete loss of β1 integrin function in vivo. In contrast, replacement of β1 integrin cytoplasmic tyrosines with phenylalanines, a mutation that prevents tyrosine phosphorylation, conserves in vivo integrin function. These results have important implications for the molecular mechanism and regulation of integrin function.

April 15, 2006

Regulation of blood and lymphatic vascular separation by signaling proteins SLP76 and Syk. (Science. 299:247)

January 10, 2003

Lymphatic vessels develop from specialized endothelial cells in preexisting blood vessels, but the molecular signals that regulate this separation are unknown. Here we identify a failure to separate emerging lymphatic vessels from blood vessels in mice lacking the hematopoietic signaling protein SLP-76 or Syk. Blood-lymphatic connections lead to embryonic hemorrhage and arteriovenous shunting. Expression of slp-76 could not be detected in endothelial cells, and blood-filled lymphatics also arose in wild-type mice reconstituted with SLP-76-deficient bone marrow. These studies reveal a hematopoietic signaling pathway required for separation of the two major vascular networks in mammals.

Rap1A positively regulates T cells via integrin activation rather than inhibit lymphocyte signaling. (Nature Immunology. 3:251)

April 2002

T cell receptor (TCR) stimulation activates the small GTPase Rap1A, which is reported to antagonize Ras signaling and induces T cell anergy. To address its role in vivo, we generated transgenic mice that constitutively expressed active Rap1A within the T cell lineage. We found that active Rap1A did not interfere with the Ras signaling pathway or antagonize T cell activation. Instead of anergy, the T lymphocytes that constitutively expressed active Rap1A showed enhanced TCR-mediated responses, both in thymocytes and mature T cells. In addition, Rap1A activation was sufficient to induce strong activation of the beta1 and beta2 integrins via an avidity-modulation mechanism. This shows that, far from playing an inhibitory role during T cell activation, Rap1A positively influences T cells by augmenting lymphocyte responses and directing integrin activation.

Identification of a cross-reactive self ligand in virus-mediated autoimmunity. (Eur. J. Immunol. 29:2886)

March 28, 1999

Molecular mimicry has been considered to be one of the potential mechanisms underlying the induction of autoimmune diseases. Using a TCR‐transgenic model specific for lymphocytic choriomeningitis virus (LCMV) we have examined the potential for cross‐reactive recognition of tissue‐restricted self peptides. Several peptides were identified that were able to cross‐react with the TCR‐transgenic virus‐specific T cells in vitro. One peptide was derived from dopamine β‐mono‐oxygenase, an enzyme expressed in the adrenal medulla. Interestingly, after activation of the transgenic T cells with LCMV glycoprotein peptides or viruses, infiltration of the adrenal medulla was detected in conjunction with alterations in dopamine metabolism. However, complete destruction of the adrenal medulla was not observed. This suggests that molecular mimicry may be sufficient for self recognition and infiltration, but other factors clearly contribute to chronic autoimmune disease.

Peptide-induced positive selection of TCR transgenic thymocytes in a coreceptor independent manner. (Immunity. 6:643)

May 1, 1997

T cell receptor (TCR) transgenic thymocytes specific for the LCMV gp peptide are normally positively selected to the CD8 lineage. Transgenic thymocyte development was substantially reduced in the absence of these CD8 coreceptors. However, efficient positive selection was restored when TCR transgenic CD8−/− fetal thymic lobes were cultured with a peptide variant of the wild-type ligand. These mature thymocytes were functional, as shown by their ability to respond against strong peptide agonists. Additional experiments demonstrated that transgenic positive selection was peptide-specific. These results prove that CD8 does not possess essential signaling properties that are necessary for T cell development. In addition, the unilateral commitment of transgenic thymocytes to mature CD4−TCRhi T cells expressing intracellular perforin suggests that there must be some instructive component to CD4 down-regulation and lineage commitment during thymocyte selection.

Mature T cell reactivity altered by a peptide agonist that induces positive selection. (J. Exp. Med. 183:1093)

March 1, 1996

Recent studies have investigated how defined peptides influence T cell development. Using a T cell receptor-transgenic beta2-microglobulin-deficient model, we have examined T cell maturation in fetal thymic organ cultures in the presence of various peptides containing single-alanine substitutions of the strong peptide agonist, p33. Cocultivation with the peptide A4Y, which contains an altered T cell contact residue, resulted in efficient positive selection. Several in vitro assays demonstrated that A4Y was a moderate agonist relative to p33. Although A4Y promoted positive selection over a wide concentration range, high doses of this peptide could not induce clonal deletion. Thymocytes maturing in the presence of A4Y were no longer able to respond to A4Y, but could proliferate against p33. These studies demonstrate that (a) peptides that induce efficient positive selection at high concentrations are not exclusively antagonists; (b) some agonists do not promote clonal deletion; (c) positive selection requires a unique T cell receptor-peptide-major histocompatibility complex interaction; and (d) interactions with selecting peptides during T cell ontogeny may define the functional reactivity of mature T cells.

Tumor necrosis factor receptor p55 mediates deletion of peripheral cytotoxic T lymphocytes in vivo. (Eur. J. Immunol. 26:3055)

December 1996

Cellular death of activated lymphocytes down‐regulates immune responses and is involved in maintaining self tolerance. Signals associated with ligation of the membrane molecule Fas lead to lymphocyte apoptosis, but additional, Fas independent mechanisms have been postulated. Here, we show a marked expansion and prolonged persistence of functional activated cytotoxic T cells in mice lacking the tumor necrosis factor (TNF) receptor p55. In the absence of this receptor, peripheral lymphocyte apoptosis was significantly reduced in vivo. The prolonged thymocyte survival was associated with functional anergy, since the T cells no longer proliferated in vitro when stimulated with peptide antigen. However, specific cytotoxic effector function was easily detected in vitro. We conclude that the TNF receptor p55 is involved in peripheral T cell deletion in vivo.

T cell responses are governed by avidity and costimulatory thresholds (Eur. J. Immunol. 26:2017)

September 1996

We analyzed the avidity and CD28-mediated co-stimulatory requirements for the activation of T cells in vivo and in vitro. The strength of the T cell/antigen-presenting cell interaction was varied by using altered peptide ligands for stimulation. Co-stimulatory requirements were studied using T cells from CD28-deficient mice. The results indicate that T cell activation is not an all-or-nothing event, but occurs in distinct steps. For each step, a certain avidity, co-stimulatory threshold or both, must be met. Depending upon the strength of the interaction between the T cell receptor and the major histocompatibility complex/peptide and the presence of CD28 co-stimulatory signals, T cells may undergo blast formation alone or proliferate or eventually both proliferate and differentiate to effector cells. Thus, T cell activation is governed by both avidity and co-stimulatory thresholds.

Duration of TCR stimulation determines costimulatory requirements of T cells. (Immunity. 5:31)